The present invention generally relates to the use of thioredoxin as, inter alia, a cell growth stimulator, as well as a screen for agents that are useful in reducing or eliminating thioredoxin-associated apoptosis inhibition and agents that are useful in inhibiting thioredoxin stimulated cell growth.
Thioredoxin is a low molecular weight (Mr 11,000-12,000) redox protein found in both prokaryotic and eukaryotic cells. (Holmgren A., J. Biol. Chem., 264:13963-13966, 1989), that undergoes reversible thiol reduction by the NADPH-dependent enzyme thioredoxin reductase. Human thioredoxin, which has 5 cysteine (Cys) residues, is a 11.5 kDa protein with 27% amino acid identity to E. coli thioredoxin. Human thioredoxin contains 3 additional Cys residues not found in bacterial thioredoxin that give it unique biological properties. (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994). Cys32 and Cys35 are the conserved catalytic site cysteine residues that undergo reversible oxidation to cystine. Cys92, Cys69 and Cys73 are found in mammalian but not in bacterial thioredoxins. Cys73 appears to be particularly important for maintaining the biological activity of thioredoxin in an oxidizing environment. Thioredoxin reduces a variety of intracellular proteins including enzymes such as ribonucleotide reductase which is important for DNA synthesis, and critical Cys residues in transcription factors such as NF-xcexaB, AP-1 and the glucocorticoid receptor, thus, altering their binding to DNA. In addition to its intracellular actions, human thioredoxin has remaskable eztracellular celm growth stimulating properties. It has been reported (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994) that thioredoxion is identical to a growth factor reported to be secreted by human HTLV-1 transformed leukemia cell lines (Fox J A, et al., Proc.Natl. Accd. Sci. USA, 84:2663-2677, 1987). It has also been found that human recombinant thioredoxin will stimulate the growth of a wide variety of fibroblast and human solid tumor cell lines in culture (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995; Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994). E. coli thioredoxin does not stimulate cell proliferation.
Thioredoxin was first studied for its ability to act as reducing co-factor for ribonucleotide reductase, the first unique step in DNA synthesis. (Laurent T C, et al., 15 J. Biol. Chem., 239:3436-3444, 1964). More recently thioredoxin has been shown to exert redox control over a number of transcription factors modulating their binding to DNA and thus, regulating gene transcription. Transcription factors regulated by thioredoxin include NF-xcexaB (Matthews J R, et al., Nucl. Acids Res., 20:3821-3830, 1992), TFIIIC (Cromlish J A, et al., J. Biol. Chem., 264:18100-18109, 1989), BZLFI (Bannister 20 A J, et al., Oncogene, 6:1243-1250, 1991), the glucocorticoid receptor (Grippo J F, et al., J. Biol. Chem., 258:13658-13664, 1983) and, indirectly through a nuclear redox factor Ref-1/HAPE, thioredoxin can regulate AP-1 (Fos/Jun heterodimer) (Abate C, et al., Science 249:1157-1161, 1990). Thioredoxin is also a growth factor with a unique mechanism of action.
Human thioredoxin has been sequenced and cloned. (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994; Deiss L P, et al., Science 252:117-120, 1991). It has been shown that the deduced amino acid sequence of thioredoxin is identical to that of a previously known protein called eosinophil cytotoxicity stimulating factor (Silberstein D S. et al. J. Biol. Chem. 268:9138-9142, 1993) or adult T-cell leukemia-derived factor (ADF) (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994). ADF has been reported to be secreted by virally transformed leukemic cell lines and to stimulate their growth (Yodoi J, et al., Adv. Cancer Res., 57:381-411, 1991). These observations have been extended to show that human recombinant thioredoxin stimulates the proliferation of both normal fibroblasts and a wide variety of human solid and leukemic cancer cell lines. (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995); Powis G, et al., Oncol. Res., 6:539-544, 1994; Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994). It has been shown that thioredoxin stimulates cell proliferation by increasing the sensitivity of the cells to growth factors secreted by the cells themselves. (Gasdaska J R, et al., Cell Growth Differ., 10 6:1643-1650, 1995).
Recombinant modified thioredoxins, otherwise called mutant thioredoxins, have been developed, but no indications of uses were known in the art for any particular mutant form. In a wild type thioredoxin, the cysteine (Cys) residues at the conserved -Cys32-Gly-Pro-Cys35-Lys active site of thioredoxin undergo reversible oxidation-reduction catalyzed by the NADPH-dependent flavoprotein thioredoxin reductase. (Luthman M, et al., Biochem., 21:6628-6633, 1982). It has been reported that mutation of the active site Cys32 and Cys35 residues to serine (Ser) residues, either singly or together (C32S/C35S thioredoxin), results in a compound that is redox inactive (i.e., it is not a substrate for reduction by thioredoxin reductase) and that does not stimulate cell proliferation (Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994).
Thioredoxin mRNA has been found to be over expressed by some human tumor cells (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994; Grogan T, et al., Cancer Res., 1997, in press) and since it is secreted from cells by a leaderless secretary pathway (Rubartelli A. et al., J. Biol. Chem., 267:24161-24164, 1992) it could be a growth factor for some human cancers (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995). However, the mechanism for cell growth stimulation by thioredoxin mRNA has been examined and found not to promote cell growth. Recombinant human thioredoxin is not taken up by cells and does not bind to high affinity cell surface receptors but appears to enhance the sensitivity of cells to endogenously produced or other growth factors, a mechanism termed voitocrine (Greek, voithos =helper) (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995).
The in vitro cell growth stimulating activity of human thioredoxin has been previously reported for human lymphoid and solid tumor cancer cells (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995; Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994) and for mouse fibroblast cells (Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994). The production of a Cys73xe2x86x92Ser mutant thioredoxin has been previously reported. In one study it did not act like wild-type thioredoxin as a component of a complex cell growth stimulating factor called xe2x80x9cearly pregnancy factorxe2x80x9d (Tonissen K, 10 et al., J. Biol. Chem., 268:22485-22489, 1993). In another study it was reported that Cys73xe2x86x92Ser mutant thioredoxin did not form a dimer, but cell growth stimulating activity by the mutant thioredoxin was not investigated in this study (Ren X, et al., Biochem., 32:9701-9705, 1993). However, the ability of the Cys73xe2x86x92Ser mutant and other mutant thioredoxins to stimulate cell proliferation has not been reported There have been no prior reports of administration of mutant thioredoxins in vivo.
It has been known that certain human tumor cells were found to over-express thioredoxin mRNA compared to normal lung tissue from the same subject. (Gasdaska P Y, et al., Biochem. Biophys. Acta., I 218:292-296, 1994). It has also been known that human primary colorectal tumors have exhibited elevated levels of thioredoxin mRNA compared to normal colonic mucosa (Berggren M, et al., Anticancer Res., 16:3459-3466, 1996). It has not been known that thioredoxin protein was present in certain human tumor cells, and it has not been known that thioredoxin protein played any role in preventing or enhancing tumor cell growth.
While thioredoxin itself is known, its use in identifying agents that inhibit cell growth stimulated by thioredoxin has not been previously shown.
Human thioredoxin reductase has been characterized as a protein (Oblong J E, et al., Biochem., 32:7271-7277, 1993). In addition, the general properties and the cDNA base sequence of human thioredoxin reductase is known in the art. However, it has not been disclosed or suggested in the art that thioredoxin reductase be used as an anti-tumor drug target.
The myelodysplastic syndromes (MDS) are a heterogeneous class of life threatening diseases characterized by ineffective hematopoiesis and progressive, reractory cytopenia (List A F, et al., J. Clin. Oncol., 8:1424-1441, 1990). Transformation to acute leukemia may occur in one-third of the patients, The underlying defect is decreased multilineage progenitor cell growth associated with decreased sensitivity to growth factor stimulation (Merchav S, et al., Leukemia, 5:340-346, 1991). Very high doses of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human granulocyte colony stimulating factor (G-CSF) can ameliorate neutropenia but do not improve red blood cell or platelet function (List A F, et al., J. Clin. Oncol., 8:1424-1441, 1990). Although IL-3 displays multilineage progenitor stimulatory effects in normal marrow clinical trials have shown limited ability to improve hematopoiesis in MDS (List A F, et al., Blood, 82 (Suppl. 1):377a, 1993). Thus, current treatment for MD is limited by the ability of cytokines to stimulate hematopoietic progenitor cells and the decreased sensitivity of these cells to growth factors.
The present invention relates to the use of thioredoxin as, inter alia, a cell growth stimulator, as well as a screen for agents that are useful in reducing or preventing thioredoxin-associated apoptosis inhibition in tumor cells and agents that are useful in inhibiting thioredoxin stimulated growth of tumor cells.
A non-limiting embodiment of the invention involves a method of inhibiting tumor cell growth in a tumor cell that over-expresses thioredoxin comprising contacting said tumor cell with a cell growth inhibiting effective amount of an inhibitor of thioredoxin expression. Such agents can include, inter alia, small molecular compounds that complex with and interfere with the biological action of thioredoxin, preferably those that complex with active Cys residues, antisense inhibitors of thioredoxin expression, antibodies, or inhibitors of nucleic acid expression.
A further non-limiting embodiment of the invention involves a method of reducing inhibition of apoptosis in tumor cells that over-express thioredoxin comprising contacting said tumor cells with an effective amount of an agent that inhibits thioredoxin. Such agents can include, inter alia, antibodies to this redoxin, compounds that inhibit the activity of this redoxin, preferably those that inhibit the activity of active Cys residues in the protein, cross-linking agents and the like.
A further non-limiting embodiment of the invention involves a method of identifying an agent that inhibits tumor cell growth in cells that over-express thioredoxin comprising measuring thioredoxin expression or activity in a first sample of said cells; contacting a second sample of said cells with an agent to be tested; measuring expression or activity of thioredoxin in said second sample; comparing expression or activity of thioredoxin in said first sample and said second sample; whereby a decrease in expression or activity of thioredoxin in said second sample is indicative of an agent that inhibits tumor cell growth.
A further non-limiting embodiment of the invention involves a method of identifying an agent that reduces inhibition of apoptosis in a tumor cell that over-expresses thioredoxin comprising measuring thioredoxin expression or activity in a first sample of said cells; contacting a second sample of said cells with an agent to be tested; measuring expression or activity of thioredoxin in said second sample; comparing expression or activity of thioredoxin in said first sample and said second sample; whereby a decrease in expression or activity of thioredoxin in said second sample is indicative of an agent that reduces inhibition of apoptosis.
A further non-limiting embodiment of the invention involves a method of identifying an agent that reduces thioredoxin induced inhibition of apoptosis in a tumor cell growth.
A further non-limiting embodiment of the invention involves a method of stimulating cell growth comprising introducing a nucleic acid encoding a human thioredoxin having Ser at amino acid reside 73 under conditions whereby said nucleic acid is expressed.
A further non-limiting embodiment of the invention involves a composition comprising an agent that is useful in reducing or eliminating thioredoxin-associated apoptosis inhibition and an acceptable carrier.
A further non-limiting embodiment of the invention involves a composition comprising an agent that is useful in inhibiting thioredoxin stimulated cell growth and an acceptable carrier.
The present invention is based, at least in part, on the discovery that thioredoxin protein is over-expressed in certain human tumor cells; that thioredoxin stimulates the growth of cancer cells; that thioredoxin inhibits apoptosis; that thioredoxin is over-expressed in some human primary tumors and is correlated with increased tumor cell growth and decreased apoptosis; and that agents that inhibit thioredoxin also have anti-tumor activity.
The present invention involves the new uses of thioredoxin, thioredoxin reductase, and mutant forms of thioredoxin for use in screening for anti-tumor agents. It has not been known in the art to use thioredoxin and/or thioredoxin reductase in a screening assay for anti-thioredoxin and/or anti-thioredoxin reductase agents for use as anti-tumor compounds.
The present invention further relates to the use of thioredoxin and/or thioredoxin reductase antibodies for use as anti-tumor agents.
The present invention further relates to the use of anti-sense thioredoxin or anti-sense thioredoxin reductase compounds for use as anti-tumor agents.
The present invention further relates to the use of thioredoxin nucleic acid probes and/or thioredoxin antibodies in a diagnostic assay for certain cancers.
The present invention further relates to the use of thioredoxin as a target for agents to be used in combination with existing and new treatment therapies, such as drugs and radiation, that reduce or prevent the thioredoxin-induced inhibition of apoptosis in tumor cells or to increase the sensitivity of tumor cells to these modalities.
In addition, mutant forms of thioredoxin provide proteins with additional growth stimulating activity.
These and still further objects as shall hereinafter appear are readily fulfilled by the present invention in an unexpected manner as will be readily discerned from the following detailed description of the preferred embodiments of the invention, especially when read in conjunction with the accompanying drawings.
In contrast to the present invention, none of the above cited references teach or suggest the use of thioredoxin protein according to the claimed invention.